Amyloids are filamentous polymers of aberrantly folded proteins distinguished by their cross-beta structures. Accumulation of amyloid is associated with approximately 20 human diseases, including Alzheimer's, type-2 diabetes, and rheumatoid arthritis. Amyloids fall into two broad categories: infectious and non-infectious. Infectious amyloids are called prions. We started studying yeast prion structures in 1998, focusing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. With this model, it envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for other amyloid fibrils, including amylin, and growing support for models of this kind is appearing in the scientific literature. Ongoing work is aimed at testing and refining this model; investigating fibril polymorphism; and relating amyloids to native conformations. In FY14 we focused on two projects. (1) Polyglutamine-rich amyloids. Polyglutamine (polyQ) sequences are found in a variety of innocuous proteins, but at least nine human proteins containing polyQ runs of longer than 20 residues are associated with severe hereditary neurodegenerative diseases, including Huntingtons disease, spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxia. The disease-related proteins are not sequence-similar to each other outside of the polyQ regions. The pathogenic mechanisms of these diseases are not fully understood but one thing is definitethe brain tissues of patients contain deposits of the polyQ-rich proteins. Furthermore, longer polyQ tracts (>30 residues or so) have a stronger predisposition to aggregate, leading to a higher risk and earlier onset of disease. The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. To this end, we designed polyglutamines with a few lysine's inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of beta-strands. One was 33 amino acids long (polyQKd-33) and the other has one fewer glutamine (polyQKd-32). Both were found to form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-beta structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide led to substantially different amyloid structures. PolyQKd-32 fibrils were consistently 1020% wider than polyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy (STEM). STEM analysis further revealed that the polyQKd-32 fibrils have 50% higher mass-per-length than polyQKd-33 fibrils. This distinction can be explained by a superpleated beta-structure model for polyQKd- 33 and a model with two beta-solenoid protofibrils for These data provide evidence for beta-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design. This study was recently published (1). 2) Alfa-synuclein-containing amyloids. Parkinsons disease (PD) is a chronic and progressive neurodegenerative disease affecting motor function. PD is characterized by dopaminergic neuronal cell death and by the presence of Lewy bodies. Alfa-synuclein (aS) fibrils are the main component of Lewy bodies, and previous research suggests that its fibrillation is part of the disease pathology. Normally, the 140 aa long protein has a membrane remodeling function which we are also researching, as reported in project AR027015-19. aS is alfa-helical when associated with lipid and a random coil in solution. In fibril formation, the protein polymerizes into a cross-beta structure. Despite their high clinical relevance, structural information on aS-containing amyloid fibrils has been elusive and such information is the goal of this project. Recombinant aS was expressed in E. coli, purified and assembled into fibrils, which were observed by cryo-EM in our laboratory and by dark-field STEM at the Brookhaven STEM facility. The resulting cryo-EM images revealed that aS fibrils are 5 nm in diameter and more or less straight, and they have an axial repeat of 75 nm. Mass-per-length measurements made from the STEM data gave a unimodal distribution with a mean density equivalent to two subunits per 0.47 nm axial rise. This suggests that the fibril is in fact a pair of protofibrils - each with a parallel superpleated structure - wrapping around a common axis. Reconstruction of the cross-section indicates that each protofibril has two components. These observations, which afford a basis for modeling studies, are currently being prepared for publication.